PROGRESS IN NATURAL SCIENCE V ol .15 , No .3 , M arch 2005 Phylogenetic studies of Chinese labeonine fishes (Teleostei : Cyprinidae)based on the mitochondrial 16S rRNA gene＊ LI Junbing1, 2 , WANG Xuzhen1 , HE Shunping1 ＊＊ and CHEN Yiy u1 (1 .Institute of Hydrobiology , Chinese Academy of Sciences, Wuhan 430072 , China;2 .Graduate School of the Chinese Academy of S ciences, Beijing 100039 , C hina) Received July 15 , 2004 ;revised November 11 , 2004 Abstract The mitochondrial 16S ribosomal RNA gene is sequenced from 24 ingroups taxa, including 18 species from Labeoninae grouped in 13 genera .Phylogenetic analyses are subjected to neighbor joining, maximum parsimony , maximum likelihood and Bayesian analyses.Phylogenetic analysis indicates that Labeoninae is basically a monophyletic assem blage and can be divided into 2 major clades:one comprising the genera Cirrhin us, Crossocheilus and Garra ;and the other consisting of the genera Labeo , Sinilabeo , Osteochilus , Pseu- doorossocheilus, Parasinilabeo , Ptychidio , S emilabeo , Pseudogyricheilus, Rectori and Discogobio .According to our present analysis, the features such as the presence of the adhesive disc on the chin and the pharyngeal teeth in 2 row s used in the traditional taxonomy of Labeoninae provide scarce information for phylogeny of labeonine fishes. Keywords: 16S rRNA, Labeoninae, monophyly , phylogeny , Cyprinidae. Labeoninae , one of the subfamilies contained species of labeonine fishes , so they cannot be used as diag- w ithin Cy prinidae , is a huge g roup including more nostic for species otherwise than garrini group[ 3] . than 300 species in approximately 26 genera .Labeo- nine fishes are hig hly adaptive to torrential habitats , Based on osteological synapomorphy , Chen et al . treated Labeoninae as a monophylotic assemblage within and they are mainly distributed in the tropical and [ 5] subtropical regio ns of Africa and Asia[ 1] .The labeo- Cyprinidae , and placed Labeoninae in Barbini Series . nine know n in Africa is a diversified g roup and com- Because of the increase of the new records of species and its important role in economy , Labeonins has received prises about 120 species and subspecies placed in 2 [ 6—8] genera , Labeo and Garra[ 2] .This assemblage in- considerable taxonomical attention .However , all the cludes 20 genera over 60 species in China , w ithin previous phylogenies were focused on a fraction of labeo- w hich there are 8 genera endemic to China[ 1] .In nine species only .Therefore , up to date no subsequent China , labeonine fishes are mainly distributed in the work has been done to provide phylogenetic information w ater systems in south of the Yang tze River .The on the taxa of this group . subfamilies Labeoninae and Barbininae , together with The mitocho ndrial DNA has been w idely used in most of the genera of Danioninae and the genus Puntio- [ 1] the studies of molecular sy stematics of verte- plites of Cyprininae , constitute the south Asian g roup . brates[ 9, 10] , and the mitochondrial 16S rRNA gene Labeonins had a taxonomic difficulty because of sequence w as used as an effective genetic mark- [ 11 , 12] the lack of appropriate taxonomic features for assigna- er .In this study , we used the sequences of the tion of these included genera to higher taxonomic cat- mitochondrial DNA 16S rRNA fo r 18 labeonine egories.Labeonine fishes were affiliated to the sub- species to test the monophyly of Labeoninae , and we family Barbininae[ 3] .Based on mo rphology of barbels also aimed to construct phy logenetic relationships of and the pattern of innervations correlated w ith bar- labeonine fishes at the genus level . bels , How es placed labeonins into the subfamily Cyprininae[ 4] .On account of the peculiar morphology 1 Materials and methods of lips and associated structures adapted for torrential 1 .1 Sam ple collection inhabit and the pharyngeal teeth in 2 row s , this as- semblage w as recog nized as Garrinae .These two A total of 27 species , including 18 species in 13 characters are , however , irregularly distributed among the genera from Labeoninae and the others from the sum- ＊ Supported National Natural S cience Foundation of China and C hinese Academy of Sciences (Grant Nos:30225008 & kscxz-sw-101B) ＊＊ To w hom correspondence should be addressed .E-mail:clad @ihb .ac.cn 214 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .3 2005 family Schizo thoracinae , Barbininae and Cy prininae , tions of origin and other relevant info rmation are giv- w ere used for this study .The data of Cyprinus en in Table 1 .All the tissues used for ex traction of carpio (NC001606 ) and Carassius auratus DNA were preserved in 95 % ethanol and most of (NC002079)w ere retrieved from GenBank .My xo- them w ere deposited in the Freshw ater Fish M useum cyprinus asiaticus w as chosen as the outg roup ow ing of Institute of Hydrobiology , Chinese Academy of to its clear relationship w ith family Cy prinidae .Loca- Sciences . Table 1 . Species identifications, locations of origin and the length of analyzed sequence Taxons Species Location Length (bp) Labeoninae Cirrhin us molitorella Tengxian , Guangxi 1208 Osteoch ilus sa lsburyi Rongan , Guangxi 1211 Sinilabeo laticeps Mengla , Yunnan 1176 Crossocheilus latius Tengchong , Yunnan 1215 Labeo yu nnanensis Mengla , Yunnan 1217 Pseudoorossocheilus bamaensis Tiane, Guangxi 1243 Parasin ilabeo assinilabeo Rongan , Guangxi 1182 Ptych idio jord ani Tiane, Guangxi 1144 Semilabeo notabilis Tiane, Guangxi 1121 Pseudogyrincheilus procheilus Luding , Sichuan 1274 Rectoris posehensis Duan , Guangxi 1258 Discogobio la ticeps Tiane, Guangxi 1149 Discogobio bismargaritus Tiane, Guangxi 1157 Discogobio brachyphysa llidos Jinxiu , Guangxi 1148 Discogobio tetrabarbatus Rongan , Guangxi 1271 Garra pingi hainanensis Changhua, Ledong 1174 Garra kempi Chayu Ⅴ , Tibet 1149 Garra orientails Changhua, Ledong 1229 Barbininae Onychostoma sima Hejiang , Sichuan 1254 Spinibarbus holland i Tunxi, Huangshan 1148 Cyp rininae Procyp ris rabaudi Hejiang , Sichuan 1215 Carassius au ratus NC 002079 1839 Cyp rin us carpio NC 001606 1839 Cyp rin us m ultitaen iata Guiping , Guangxi 1234 Schizothoracinae Schizothora x walton i Chayu Ⅳ , Tibet 1341 Schizothora x molesworthi Chayu Ⅴ , Tibet 1247 Family Catostomidae Myxocyp rin us asiaticus Wuhan , Hubei 1193 1 .2 DNA ex traction , PCR amplification and se- ing agarose gels , recovered from the gels and purified quencing using Biostar Glassmilk DNA purification Kit follow- ing the manufacture' s instructions .The purified frag- Total DNA w as extracted from muscle tissues o r ments were directly sequenced in both forw ard and fins follow ing Ausubel' s method[ 13] .The follow ing reverse directions by Shang hai Biostar Genechip Inc . primers , 16S L (5′- CTT ACA CCG AGA ARA CAT 1 .3 Sequence analy sis C -3′)and 16SH (5′- CTT AAG C TC CAA AGG GTC -3′), were used fo r amplification and sequenc- Multiple alignments of sequences were performed ing .Amplification reactions w ere performed in a using Clustal W[ 14] with a gap-opening penalty of 20 60 μL volume containing approximately 100 ng of and a gap-extension penalty of 20 and Seaview[ 15] template DNA , 0 .75 μL dNTP (2 .5 mmol/L of alignment edito r and verified by eye .Measures of nu- each), 1 .5 μL of each primer , 5 μL of 10 X reaction cleotide composition were carried out by MEGA2 .1 buffer and 3 units of Taq DNA polymerase software[ 16] .The mutational saturation for nucleotide (Biostar).The thermocycling conditions included an substitutions w as examined by plotting transversions initial denaturation at 95 ℃ for 3 min , followed by 30 and transitions against Kimura two-parameter dis- cycles of 94 ℃ for 30 s , 60 ℃ for 30 s , and 72 ℃ fo r tance using DAM BE4 .1[ 17] ;saturation w as estimated 1 min and were completed w ith a final extension at by the extent to which the slope of a linear regression 72 ℃ for 5 min .The amplified fragments were frac- departs from a value of 1 .Aligned sequences w ere an- tionated by electrophoresis through 0 .8 % low-melt- alyzed using maximum parsimony (M P), neighbor Prog ress in Natural Science Vol.15 No .3 2005 w ww .tandf.co .uk/journals 215 joining (NJ), m aximum likelihood (M L ) and age proportions of the different nucleotide was T = Bayesian methods .All phylogenetic analyses includ- 0 .201 , C =0 .23 , A =0 .38 , G =0 .19 .These data ing maximum parsimony , neighbo r joining and maxi- clearly show that the base composition is somew hat mum likelihood were performed w ith PAUP A-rich and the base pair composition is rich in A and 4 .0b10[ 18] except for Bayesian method , the latter w as T bases (58 .1 %).An overall transitions/transver- carried out with M rBayes3 .0b4[ 19] . sions ratio observed w as 2 .1 for these data sets .The saturation of transition and transversion changes was M aximum parsimony search w as conducted using checked by plotting the percentage of sequence diver- heuristic search methods w ith tree bisection-reconnec- gence against the pairwise number of inferred substi- tion (TBR)branch sw apping , and 10 random se- tutions .The transitions/ transversions ratio did not quence addition replicates .Non-parametric bootstrap exhibit any plateau and indicated a non-mutational analy ses w ith 1000 pseudoreplicates and 10 random saturation (data no t show n).The uncorrected p-dis- sequence additions w ere conducted on the equally tance matrix observed from the analysis of the align- w eighted data .In order to determine w hich model is ment of all sequences is also not show n here , and the the best-fit , w e selected models using Modeltest value of pairw ise distance among labeonine fishes [ 20] 3 .06 .Finally , the GTR model w hich incorporates ranged from 0 .1 % to 16 %, and from 14 .8 % to rate variation (Γ)along w ith the number of invariable 20 .4 % betw een ingroups and outg roup .